The membrane-bound enzyme system which causes acidification of the medium upon oxidation through a critical voltage range above 300mV has been found to operate in beef heart mitochondria. An essential component of the reducting power in the E. coli system has been identified as a charge transfer complex involving an amine and a quinone. The source of protons has been identified as water. A powerful new technique for resolving a complex series of spectra into individual spectra and the quantitative and thermodynamic properties of the individual components such as redox potentials, number of electrons transferred in an oxidation and pK's for acids has been developed and tested.